Sepsis is a life-threatening bloodstream infection that results in significant morbidity and mortality. Prompt and appropriate therapy is critical to improved outcomes, but determining the infectious micro-organism can take days. Choice of therapy is further complicated by the dramatic rise in the number of infections caused by antibiotic-resistant organisms. Rapid identification of the etiological agent along with targeted resistance information would enable optimized treatment and improved patient care. The FilmArray(tm) (FA) platform developed and manufactured by BioFire Diagnostics, LLC, is well-suited to meet the challenges of rapid sepsis diagnosis. It is a highly multiplexed, easy to use, sample to result PCR- based diagnostic system. In this project, we propose to develop and optimize a direct from blood FA panel to detect important nosocomial bacteria and their principal genetic determinants of antibiotic resistance. We will incorporate key additional technologies for optimal sensitivity and specificity. This project involves four Aims: Specific Aim 1: Develop and Optimize the FA Sepsis Direct Panel We will develop and optimize a panel for the FA system to simultaneously detect bacteria causing nosocomial infection, as well their primary determinants of antibiotic resistance. This panel will target at least 12 different Gram-negative and Gram-positive bacteria as well as 16 antibiotic resistance genes. Specific Aim 2: Develop External Enrichment Procedure of Bacteria from Blood We will develop and optimize a selective lysis and centrifugation protocol for the concentration of bacterial pathogens from large volumes of blood. With this protocol we will concentrate the viable bacteria present in up to 10 mL of whole blood to a 0.1 mL volume that can be completely introduced into a FA pouch for analysis. Specific Aim 3: Reduce Impact of Bacteria Genomic DNA (bgDNA) Background in the FA pouch We will reduce the impact of bgDNA in FA pouches by 1) working with our raw materials suppliers to lower the amount of bgDNA entering the FA pouch production line and 2) mature technology to modify sample nucleic acid sequences to create non-canonical sequences that can be recognized by unique PCR primers, effectively nullifying the impact of bgDNA. Specific Aim 4: Preclinical Evaluation of the FilmArray Sepsis Direct System in Whole Blood We use the assay system developed in specific aims 1 through 3 to test 1,000 whole blood samples from pediatric and adult patients with suspected sepsis in a preclinical evaluation. We will compare the results of FA Sepsis Direct panel testing to clinical and laboratory data for each patient.